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Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells.

Lizunov VA, Matsumoto H, Zimmerberg J, Cushman SW, Frolov VA - J. Cell Biol. (2005)

Bottom Line: Glucose transport in adipose cells is regulated by changing the distribution of glucose transporter 4 (GLUT4) between the cell interior and the plasma membrane (PM).This slow release of GLUT4 determined the overall increase of the PM GLUT4.It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Glucose transport in adipose cells is regulated by changing the distribution of glucose transporter 4 (GLUT4) between the cell interior and the plasma membrane (PM). Insulin shifts this distribution by augmenting the rate of exocytosis of specialized GLUT4 vesicles. We applied time-lapse total internal reflection fluorescence microscopy to dissect intermediates of this GLUT4 translocation in rat adipose cells in primary culture. Without insulin, GLUT4 vesicles rapidly moved along a microtubule network covering the entire PM, periodically stopping, most often just briefly, by loosely tethering to the PM. Insulin halted this traffic by tightly tethering vesicles to the PM where they formed clusters and slowly fused to the PM. This slow release of GLUT4 determined the overall increase of the PM GLUT4. Thus, insulin initially recruits GLUT4 sequestered in mobile vesicles near the PM. It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM.

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Confocal microscopy and TIRFM of isolated white adipose cells. (A) Differential interference contrast image of an isolated cell slightly squashed between two coverslips with nuclear region facing to the left side. (B) Flat round region of PM and adjacent thin layer of cytoplasm accessible for TIRF imaging (TIRF zone). (inset) Under TIRF illumination the fluorescence excitation intensity decreases exponentially with distance from the coverslip. (C) Three-dimensional reconstruction of confocal images of a basal adipose cell transfected with GLUT4-GFP. Use red and green glasses to view this image. (D) Randomly selected part of basal adipose cell visualized with TIRF. Substantial amounts of GLUT4 vesicles are located near the PM (within 400 nm; see Materials and methods) in randomly scattered fashion. Note the variation of fluorescence intensity of vesicles due to different positions relative to the coverslip. Bars, 10 μm.
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fig1: Confocal microscopy and TIRFM of isolated white adipose cells. (A) Differential interference contrast image of an isolated cell slightly squashed between two coverslips with nuclear region facing to the left side. (B) Flat round region of PM and adjacent thin layer of cytoplasm accessible for TIRF imaging (TIRF zone). (inset) Under TIRF illumination the fluorescence excitation intensity decreases exponentially with distance from the coverslip. (C) Three-dimensional reconstruction of confocal images of a basal adipose cell transfected with GLUT4-GFP. Use red and green glasses to view this image. (D) Randomly selected part of basal adipose cell visualized with TIRF. Substantial amounts of GLUT4 vesicles are located near the PM (within 400 nm; see Materials and methods) in randomly scattered fashion. Note the variation of fluorescence intensity of vesicles due to different positions relative to the coverslip. Bars, 10 μm.

Mentions: When isolated from tissue, white adipose cells are round and their interior is filled with a large droplet of stored triglyceride (Fig. 1, A and B), leaving a thin (1–3-μm) layer of cytoplasm between the lipid droplet and the PM (Cushman, 1970; Malide et al., 2000). In our experiments, these cells, which ordinarily float in their medium, were slightly pressed against the coverslip such that a small flattened part of the PM and nearby cytoplasm (hereafter termed the “TIRF zone”) was illuminated by the evanescent wave (Fig. 1 B and Fig. S1 A, available at http://www.jcb.org/cgi/content/full.200412069/DC1). The three-dimensional distribution and dynamics of GLUT4 vesicles in the cytoplasm of basal cells was characterized by confocal and TIRFM.


Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells.

Lizunov VA, Matsumoto H, Zimmerberg J, Cushman SW, Frolov VA - J. Cell Biol. (2005)

Confocal microscopy and TIRFM of isolated white adipose cells. (A) Differential interference contrast image of an isolated cell slightly squashed between two coverslips with nuclear region facing to the left side. (B) Flat round region of PM and adjacent thin layer of cytoplasm accessible for TIRF imaging (TIRF zone). (inset) Under TIRF illumination the fluorescence excitation intensity decreases exponentially with distance from the coverslip. (C) Three-dimensional reconstruction of confocal images of a basal adipose cell transfected with GLUT4-GFP. Use red and green glasses to view this image. (D) Randomly selected part of basal adipose cell visualized with TIRF. Substantial amounts of GLUT4 vesicles are located near the PM (within 400 nm; see Materials and methods) in randomly scattered fashion. Note the variation of fluorescence intensity of vesicles due to different positions relative to the coverslip. Bars, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171949&req=5

fig1: Confocal microscopy and TIRFM of isolated white adipose cells. (A) Differential interference contrast image of an isolated cell slightly squashed between two coverslips with nuclear region facing to the left side. (B) Flat round region of PM and adjacent thin layer of cytoplasm accessible for TIRF imaging (TIRF zone). (inset) Under TIRF illumination the fluorescence excitation intensity decreases exponentially with distance from the coverslip. (C) Three-dimensional reconstruction of confocal images of a basal adipose cell transfected with GLUT4-GFP. Use red and green glasses to view this image. (D) Randomly selected part of basal adipose cell visualized with TIRF. Substantial amounts of GLUT4 vesicles are located near the PM (within 400 nm; see Materials and methods) in randomly scattered fashion. Note the variation of fluorescence intensity of vesicles due to different positions relative to the coverslip. Bars, 10 μm.
Mentions: When isolated from tissue, white adipose cells are round and their interior is filled with a large droplet of stored triglyceride (Fig. 1, A and B), leaving a thin (1–3-μm) layer of cytoplasm between the lipid droplet and the PM (Cushman, 1970; Malide et al., 2000). In our experiments, these cells, which ordinarily float in their medium, were slightly pressed against the coverslip such that a small flattened part of the PM and nearby cytoplasm (hereafter termed the “TIRF zone”) was illuminated by the evanescent wave (Fig. 1 B and Fig. S1 A, available at http://www.jcb.org/cgi/content/full.200412069/DC1). The three-dimensional distribution and dynamics of GLUT4 vesicles in the cytoplasm of basal cells was characterized by confocal and TIRFM.

Bottom Line: Glucose transport in adipose cells is regulated by changing the distribution of glucose transporter 4 (GLUT4) between the cell interior and the plasma membrane (PM).This slow release of GLUT4 determined the overall increase of the PM GLUT4.It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Glucose transport in adipose cells is regulated by changing the distribution of glucose transporter 4 (GLUT4) between the cell interior and the plasma membrane (PM). Insulin shifts this distribution by augmenting the rate of exocytosis of specialized GLUT4 vesicles. We applied time-lapse total internal reflection fluorescence microscopy to dissect intermediates of this GLUT4 translocation in rat adipose cells in primary culture. Without insulin, GLUT4 vesicles rapidly moved along a microtubule network covering the entire PM, periodically stopping, most often just briefly, by loosely tethering to the PM. Insulin halted this traffic by tightly tethering vesicles to the PM where they formed clusters and slowly fused to the PM. This slow release of GLUT4 determined the overall increase of the PM GLUT4. Thus, insulin initially recruits GLUT4 sequestered in mobile vesicles near the PM. It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM.

Show MeSH
Related in: MedlinePlus